The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid Metabolism, Appressorial Turgor Pressure, and Virulence

Procedure

7. Insect Bioassay

Discussion

Abstract

3. RT-PCR Analysis of Gene Expression

2. GFP Fusion Constructs and Expression

Perilipins were the first identified mammalian lipid-associated proteins.

Cells store lipids in droplets

4. Fluorescence Microscopy

Studies - how mammals control lipid-based energy homeostatsis that implicated proteins of PAT domain family

M. anisopliae produce MPL1 [protein with common structural elements (N-terminal β-strands and central 3 hydrophobic regions that target and anchor mouse Perilipin A to Lipid Droplets and are critical for TAG storage) as mammalian perilipin]. Also MPL1 localised to lipid droplets and modulate the rate of hydrolysis.

5.Lipid Quantification

6.Appressorial Turgor Assay

1. Gene Clonning and Deletion

Such as - perilipin that surrounds the lipid droplets

Perilipin knock-out mice- lean and resistant to obesity

gene Mpl1- important insect fungal pathogen metahizium anisopliae that has structural similarities to mammalian perilipins

BLAST showed it has a high similarity with the virulence factor CAP20 of plant fungal pathogen Colletotrichum gloeosporioides

Gene deletion- using the plasmid pGPS3Bar

Mpl1- predominantly expressed when M.anisopliae is engaged in accumulating lipids and ectopically expressed green fluoresent protein-tagged MPL1 localized to lipid droplets

MPL1 has multiple phosphorylation site. Phosphorylation by cAMP-dependant protein kinase -> conformational change that initiates lipolysis by lipase.

Full-length cDNA of Mpl1 was amplified from a library of genes expressed by M. anisopliae in insect hemolymph

primers: PerEF & PerER introduced to o introduce
BamHI sites at both end and SmaI at 5' end

GFP gene was amplified from pEGFP with primers GfpPerF and GfpPerR

Mutant M. anisopliae lacking MPL1 have:

thinner hyphae

fewer lipid droplets

Perilipin-like proteins share ancestral function. Homologues of MPL1 are only found in pezizomycotinal ascomycetes (important plant & animal pathogens).

particularly in appressoria

product was integrated into the SmaI site of plasmid pYes2Per using an in-fusion dry-down PCR cloning kit pGfpPer

decrease in total lipids

Mpl1- acts in a perilipin-like manner that suggesting an evolutionary conserved function in lipid metabolism

Determine MPL1 localizes to LDs in a fungus
lacking endogenous perilipin-like proteins, Saccharomyces cerevisiae strain INVSc1 (Invitrogen) was transformed with pGfpPer and the transformant grown overnight in SC-U medium
plus 2% raffinose

dramatically reduced indicating that lipid droplets are required for solute accumulation

linked with the reduce ability to breach insect cuticle so that MPl1 is a pathogenecity determinant

cultures were collected by filtration and washed
three times with sterile distilled water.

Yeast cells expressing MPL1 were unable to make use of their stored lipids during starvation ∴ no phosphorylation mechanism for removing perilipin-like protein to assess lipid droplets.

mycelia was incubated in 10 ml of water or
water supplemented with 0.1% bean root exudate or 1% cuticle.

perilipin homologs found only in pezizomycotinal ascomycetes and occur as single copy genes

Expression of MPl1 in yeast cells, a fungus- that lacks perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions

Introduction

Eukaryotic cells contain droplets of triglycerides encased in phospholipid membranes

lipid droplets (LSD)

Yeast or Metarhizium cells were
fixed in 3% formaldehyde in PBS for 20 min

washed four times with PBS

No perilipin -> reduced appressorial turgor presure because MPL1 homolog in M. oryzae function in lipid metabolism (required to generate glycerol for turgor by water influx into appressorium) ∴ decreased virulence in insect host also ∴ potential as pest control.

considered to inert storage vessels for energy-rich fat

cells was stain with Nile Red or Bodipy for detecting neutral lipid

implicated in lipid disease, inflammation, diabetes, cardiovascular disease and liver disease

fat droplets being metabolically active, membrane encasing them have protein with wide ranging biochemical activities

phosphoric acid-vanillin method

best studied- mammalian proteins of perilipin family (called PAT family)such as

-perilipin

adipocyte differentiation-related protein

TIP47

proteins have characteristicsseries of hydrophobic sequences(thePATdomain)that facilitate localization to the surface of lipid droplets

Conidia and wild type mutant ere harvested in newly mycosed Manduca larvae. The samples were centrifuged, and the absorbance of supernatants was measured at 530 nm. Standard curve was generated

yeast studies

single colony of pYes2Per transformed yeast cells was incubated overnight in YPD. Cells harvested from different media was washed 3x and adjusted to A600=1.0 with aterile water

MPL1 is upregulated in aging fungal sectors but ↓ in degenerated fungus (↓ cAMP levels ∴ ↓ in lipid metabolism)

By coating droplets,perilipin forms a barrier that restricts the access of cytosolic lipases

Differences in lipid content between
treatments were compared using the Duncan’s analysis of variance analysis

During food deprivation,perilipin is phosphorylated by protein kinase A;the barrier function of perilipin is attenuated,andlipolysis increases

Results

in normal mice,perilipin-deficient mice have less fat,more muscle,a higher metabolic rate, and are resistant to diet-induced and genetic obesity

Expression Profile and Localization of MPL1

Protein Structure and Characteristics

Loss of Function Mutants Confirm That MPL1 Plays an Important Role in Lipid Homeostasis

The effects of perilipin also been studied in Drosophila where the lack of the perilipin homolog results in flies with less fat

MPL1 Affects Appressorial Differentiation and Fungal Virulence

perilipin-free animals,fat storage is a losing battle because hormone-sensitive lipase metabolizes fat as soon as it is made. Not surprisingly therefore, human perilipin variants are also associated with obesity or leanness

ascomycete Metarhizium anisopliae, a ubiquitous insect pathogen and biocontrol agent,produces a single mammalian perilipin homolog that designated as Mpl1 for Metarhizium perilipin-likeprotein

examine the potential involvement of MPL1 in this process,

Appressorial turgor pressure was assayed using serial solutions of PEG-8000

Individual wings were dipped in PEG solutions for 10 min, and the percentage of collapsed appressoria
was determined from 300 cells per PEG solution

Virulence of the wild type and Mpl1 was
assayed against newly emerged 5th instar larvae of M. sexta

Mpl1 gene expression and protein localization.

A, RT-PCR analysis of Mpl1 expression in mycelia cultured for 6 h in water, minimal medium (MM), Sabouraud dextrose broth (SDB), bean root exudates (RE), 1%(w/v) Manduca cuticle (Cut), or hemolymph (HE).

B, time course study of Mpl1 expression in 36-h MM culture supplemented with oleic acid (600 M).

C, time course study of Mpl1 expression in conidia germinating in SDB.

D and E, co-localization of neutral lipids and MPL1 demonstrated by NR staining of GFP-MPL1 expressing cellsin conidia (D), mycelium (E) of M. anisopliae, and a budding yeast (S. cerevisiae) cell (F), respectively. BF, brightfield microscopy. Bar 5 m.

The full open reading frame of Mpl1 encodes a protein of 183 amino acids (20.3 kDa with a predicted pI of 8.96). MPL1 is therefore only 35% the sizeof mouse perilipin A (Per A), but it has a similar overall structure and several conserved regions with its N-terminal sequence (overall sequence similarity E 1  105) (Fig. 1A).

MPL1 affects the number of lipid droplets and germling morphology.Wild type andMpl1 cells were stained with Bodipy to demonstratethe reduced numbers of lipid droplets in the mutant. A, wild type conidia;B, Mpl1 conidia; C, wild type germlings; D, comparatively thin Mpl1 germlings from 20-h MM cultures (Note, arrows show the transportation of lipid droplets to germ tube tips); E, wild type and F, Mpl1 appressoria produced 20-h post-inoculation on locust wings; G, wild type; and H, Mpl1 hyphal bodies harvested from the hemocoels of infected insects. Bar 5 m.

Quantification of lipids from Metarhizium conidia and yeast (S.cerevisiae) cells. A, wild type and Mpl1 conidia harvested from mycosed M. sexta cadavers, PDA, and PDA plus OA. B, yeast cells transformed with Mpl1 under the control of the GAL1 promoter were grown overnight in noninductive (YPD or 2% raffinose (Raf)) or inductive media (2% galactose (Gal)) with or without OA and then starved (S) in sterile water for 20 h before lipid assay. Mean lipid content for columns labeled with the same letter are not significantly different ( 0.01, Duncan’s analysis of variance analysis).

MPL1 affects appressorialdifferentiation and turgorpressure.The appressoria produced by conidia germinating on locust hind wings were visualized by staining with Calcofluor white to show the formation of septa (indicated by an arrow) between the appressoria (AP) and conidia (CO) in wild type (A) but not Mpl1 germlings (B). Relatively higher levels of Mpl1 appressoria collapse when infected locustwingsare immersed in serial solutions of PEG-8000(PEG)for 10 min (C). The insetsshow collapsed appressoria. Bar 5 m.

The survival of M. sexta larvae after infection with wild type or Mpl1 conidia. A, survival of Manduca larvae following topical application with 2  107 conidia/ml suspensions of wild type or Mpl1 strains (control insects were dipped in water). B, survival of Manduca larvae following injection into the second proleg with 10 l of 5  106 conidia/ml suspensions insects were dipped in water). B, survival of Manduca larvae following injection into the second proleg with 10 l of 5  106 conidia/ml suspensions (control insects were injected with 10 l water).